Iron Deposition after Transient Forebrain Ischemia in Rat Brain

In this study, we investigated the iron deposition in the cerebral cortex, hippocampus CA1 area and corpus striatum pars dorsolateralis in a rat model of cerebral ischemia. Forebrain ischemia was induced by four-vessel occlusion for 20 min. Using iron histochemistry, regional changes were examined from 1 to 8 weeks of postischemic recirculation. Neuronal death was demonstrated in pyramidal cells of the hippocampal CA1 area and in the dorsolateral part of the corpus striatum, which are known as areas most vulnerable to ischemia. Iron deposition in hippocampal CA1 area was coupled to delayed pyramidal cell death. Perl's reaction with DAB intensification revealed of the 1 week iron deposits in the CA1 area, which gradually increased and formed clusters by 8 weeks. In the corpus striatum, strong iron staining was observed in injured cellular layer pars dorsolateralis 1 week after recirculation. Granular iron was deposited in the cytoplasm of pyramidal cells in layers III and V of the frontal cortex after 2 weeks of recirculation. In contrast to the hippocampus and striatum, the cerebral cortex did not develop severe neuronal cell death and atrophy immediately after the ischemic insult, which suggest that the neuronal cell death in the cerebral cortex occurs extremely late.     

Hepcidin is involved in iron regulation in the ischemic brain

Oxidative stress plays an important role in neuronal injuries caused by cerebral ischemia. It is well established that free iron increases significantly during ischemia and is responsible for oxidative damage in the brain. However, the mechanism of this ischemia-induced increase in iron is not completely understood. In this report, the middle cerebral artery occlusion (MCAO) rat model was performed and the mechanism of iron accumulation in cerebral ischemia-reperfusion was studied. The expression of L-ferritin was significantly increased in the cerebral cortex, hippocampus, and striatum on the ischemic side, whereas H-ferritin was reduced in the striatum and increased in the cerebral cortex and hippocampus. The expression level of the iron-export protein ferroportin1 (FPN1) significantly decreased, while the expression of transferrin receptor 1 (TfR1) was increased. In order to elucidate the mechanisms of FPN1 regulation, we studied the expression of the key regulator of FPN1, hepcidin. We observed that the hepcidin level was significantly elevated in the ischemic side of the brain. Knockdown hepcidin repressed the increasing of L-ferritin and decreasing of FPN1 invoked by ischemia-reperfusion. The results indicate that hepcidin is an important contributor to iron overload in cerebral ischemia. Furthermore, our results demonstrated that the levels of hypoxia-inducible factor-1α (HIF-1α) were significantly higher in the cerebral cortex, hippocampus and striatum on the ischemic side; therefore, the HIF-1α-mediated TfR1 expression may be another contributor to the iron overload in the ischemia-reperfusion brain.     

Glial fibrillary acidic protein is greatly modified by oxidative stress in aceruloplasminemia brain

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by mutations in the ceruloplasmin (Cp) gene. The neuropathological hallmark of this disease is intracellular iron overload, which is thought to lead to neuronal cell death through increased oxidative stress. We evaluated and characterized protein oxidation in the brain of a patient with this disease. The protein carbonyl content in the cerebral cortex of the patient was elevated compared to controls. Furthermore, peptide mass fingerprinting and partial amino acid sequencing identified glial fibrillary acidic protein (GFAP) as the major carbonylated protein in the cerebral cortex of the patient. In conjunction with the facts that Cp mainly localizes to astrocytes in the central nervous system and that astrocytes are loaded with much more iron than neurons in the cerebral cortex, our findings indicate that Cp deficiency may primarily damage astrocytes. We speculate that the dysfunction of astrocytes may be causatively related to neuronal cell loss in aceruloplasminemia.     

Glial Fibrillary Acidic Protein is Greatly Modified by Oxidative Stress in Aceruloplasminemia Brain

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by mutations in the ceruloplasmin (Cp) gene. The neuropathological hallmark of this disease is intracellular iron overload, which is thought to lead to neuronal cell death through increased oxidative stress. We evaluated and characterized protein oxidation in the brain of a patient with this disease. The protein carbonyl content in the cerebral cortex of the patient was elevated compared to controls. Furthermore, peptide mass fingerprinting and partial amino acid sequencing identified glial fibrillary acidic protein (GFAP) as the major carbonylated protein in the cerebral cortex of the patient. In conjunction with the facts that Cp mainly localizes to astrocytes in the central nervous system and that astrocytes are loaded with much more iron than neurons in the cerebral cortex, our findings indicate that Cp deficiency may primarily damage astrocytes. We speculate that the dysfunction of astrocytes may be causatively related to neuronal cell loss in aceruloplasminemia.     

Glial cells contribute more to iron and aluminum accumulation but are more resistant to oxidative stress than neuronal cells

Iron (Fe) and aluminum (Al) have been implicated in the pathogenesis of Alzheimer's disease (AD). In this study, we examined neuronal and glial cells to clarify which contributes most to metal accumulation after internalization through the transferrin-independent iron uptake (Tf-IU) systems in primary neuronal and glial predominant (NP and GP) cells from rat cerebral cortex, which affect the accumulation of transition metals in a variety of cultured cells. Al more significantly upregulated the Tf-IU activity in GP cells than in NP cells. GP cells were more resistant to Fe and Al exposure than NP cells. However, a chemiluminescence analysis specific for reactive oxygen species (ROS) showed that ROS levels in Fe- or Al-loaded NP cells were twice as high as in Fe- or Al-loaded GP cells. Northern blot analysis and gel retardation assay showed that the Al and Fe exposure taken up by the cells suppress Tf receptor mRNA expression to a greater extent in GP than NP cells, indicating that Al and Fe more markedly accumulate in glial than in neuronal cells. These results suggest that glial cells rather than neuronal cells contribute to the metal accumulation and are more resistant to oxidative stress caused by metals than neuronal cells. The present study may help to explain the pathogenesis of neurodegeneration in AD disorders caused by metal-generated oxidative stress.     

Antioxidative role of selenoprotein W in oxidant-induced mouse embryonic neuronal cell death

It has been reported that selenoprotein W (SelW) mRNA is highly expressed in the developing central nerve system of rats, and its expression is maintained until the early postnatal stage. We here found that SelW protein significantly increased in mouse brains of postnatal day 8 and 20 relative to embryonic day 15. This was accompanied by increased expression of SOD1 and SOD2. When the expression of SelW in primary cultured cells derived from embryonic cerebral cortex was knocked down with small interfering RNAs (siRNAs), SelW siRNA-transfected neuronal cells were more sensitive to the oxidative stress induced by treatment of H₂O₂ than control cells. TUNEL assays revealed that H₂O₂-induced apoptotic cell death occurred at a higher frequency in the siRNA-transfected cells than in the control cells. Taken together, our findings suggest that SelW plays an important role in protection of neurons from oxidative stress during neuronal development.     

Time-Course Change of Redd1 Expressions in the Hippocampal CA1 Region Following Chronic Cerebral Hypoperfusion

Redd1, also known as RTP801/Dig2/DDIT4, is a stress-induced protein and marked changes of Redd1 expression occurs in response to hypoxia or cerebral ischemia. In the present study, we examined the time-course changes in Redd1 protein expressions in the rat hippocampal CA1 region following chronic cerebral hypoperfusion (CCH) induced by permanent bilateral common carotid arteries occlusion (2VO). Redd1 immunoreactivity in the pyramidal neurons of the hippocampal CA1 region was increased at 7 days after 2VO surgery, and then the immunoreactivity was decreased with time. Especially, very weak Redd1 immunoreactivity was observed in the hippocampal CA1 region at 28 days after 2VO surgery. Western blot analysis showed that Redd1 level in the hippocampal CA1 region was significantly increased at 7 days following CCH and significantly decreased at 28 days after 2VO surgery, compared with that of the sham-operated group. These results indicate that Redd1 expressions is markedly changed in the hippocampal CA1 region following CCH and that change of Redd1 expression may be associated with the CCH-induced neuronal damage in the hippocampal CA1 region.     

Iron and glutathione at the crossroad of redox metabolism in neurons

Neurons, as non-dividing cells, encounter a myriad of stressful conditions throughout their lifespan. In particular, there is increasing evidence that iron progressively accumulates in the brain with age and that iron induced oxidative stress is the cause of several forms of neurodegeneration. Here, we review recent evidence that gives support to the following notions: 1) neuronal iron accumulation leads to oxidative stress and cell death; 2) neuronal survival to iron accumulation associates with decreased expression of the iron import transporter DMT1 and increased expression of the efflux transporter IREG1; and 3) the adaptive process of neurons towards iron-induced oxidative stress includes a marked increase in both the expression of the catalytic subunit of gamma glutamate-cysteine ligase and glutathione. These findings may help to understand aging-related neurodegeneration hallmarks: oxidative damage, functional impairment and cell death.     

Induction of apoptotic change in the rat hippocampus caused by ferric nitrilotriacetate

Iron, a source of oxidative stress, plays a major role in the pathology of neurodegenerative disease. In Alzheimer's disease, the hippocampus is vulnerable to oxidative stress, leading to impairment in memory formation. In our previous study, a brain oxidative reaction was induced after intraperitoneal injection of ferric nitrilotriacetate (Fe-NTA). However, since only a small amount of iron reached the brain in the previous study, Fe-NTA was administered into the hippocampus using an osmotic pump in this study. After continuous injection of Fe-NTA for 2 weeks, a high level of apoptotic change was induced in the hippocampus, in accordance with the iron localization. After injection for 4 weeks, the hippocampus was totally destroyed. A small amount of iron infiltrated into the cerebral cortex and the striatum, and deposition was observed at the choroid plexus and ependymal cells. However, no apoptotic reaction or clear tissue injury was observed in these areas. In addition, muscarinic acetylcholine receptors (M1, M2, and M4) were decreased in both the cortex and hippocampus while it increased in the striatum. Thus, the hippocampus is likely vulnerable to oxidative stress from Fe-NTA, and the oxidative stress is considered to bring the disturbance in the muscarinic acetylcholine receptors.     

Transient focal cerebral ischemia down-regulates glutamate transporters GLT-1 and EAAC1 expression in rat brain

Transient focal cerebral ischemia leads to extensive excitotoxic neuronal damage in rat cerebral cortex. Efficient reuptake of the released glutamate is essential for preventing glutamate receptor over-stimulation and neuronal death. Present study evaluated the expression of the glial (GLT-1 and GLAST) and neuronal (EAAC1) subtypes of glutamate transporters after transient middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia in rats. Between 24h to 72h of reperfusion after transient MCAO, GLT-1 and EAAC1 protein levels decreased significantly (by 36% to 56%, p < 0.05) in the ipsilateral cortex compared with the contralateral cortex or sham control. GLT-1 and EAAC1 mRNA expression also decreased in the ipsilateral cortex of ischemic rats at both 24h and 72h of reperfusion, compared with the contralateral cortex or sham control. Glutamate transporter down-regulation may disrupt the normal clearance of the synaptically-released glutamate and may contribute to the ischemic neuronal death.     

Intermittent Fasting Attenuates Hallmark Vascular and Neuronal Pathologies in a Mouse Model of Vascular Cognitive Impairment

Background - Chronic cerebral hypoperfusion (CCH) is an important pathophysiological mechanism of vascular cognitive impairment (VCI). The heterogeneous effects of CCH complicate establishing single target therapies against VCI and its more severe form, vascular dementia (VaD). Intermittent fasting (IF) has multiple targets and is neuroprotective across a range of disease conditions including stroke, but its effects against CCH-induced neurovascular pathologies remain to be elucidated. We therefore assessed the effect of IF against CCH-associated neurovascular pathologies and investigated its underlying mechanisms.Methods - Male C57BL/6NTac mice were subjected to either ad libitum feeding (AL) or IF (16 hours of fasting per day) for 4 months. In both groups, CCH was experimentally induced by the bilateral common carotid artery stenosis (BCAS) method. Sham operated groups were used as controls. Measures of leaky microvessels, blood-brain barrier (BBB) permeability, protein expression of tight junctions, extracellular matrix components and white matter changes were determined to investigate the effect of IF against CCH-induced neurovascular pathologies.Results - IF alleviated CCH-induced neurovascular pathologies by reducing the number of leaky microvessels, BBB breakdown and loss of tight junctional proteins. In addition, IF mitigated the severity of white matter lesions, and maintained myelin basic protein levels, while concurrently reducing hippocampal neuronal cell death. Furthermore, IF reduced the CCH-induced increase in levels of matrix metalloproteinase (MMP)-2 and its upstream activator MT1-MMP, which are involved in the breakdown of the extracellular matrix that is a core component of the BBB. Additionally, we observed that IF reduced CCH-induced increase in the oxidative stress marker malondialdehyde, and increased antioxidant markers glutathione and superoxide dismutase. Overall, our data suggest that IF attenuates neurovascular damage, metalloproteinase and oxidative stress-associated pathways, and cell death in the brain following CCH in a mouse model of VCI.Conclusion - Although IF has yet to be assessed in human patients with VaD, our data suggest that IF may be an effective means of preventing the onset or suppressing the development of neurovascular pathologies in VCI and VaD.     

Psychological Stress Expands Low Molecular Weight Iron Pool in Cerebral Cortex,Hippocampus, and Striatum of Rats

We have previously demonstrated that psychological stress (PS) can cause iron to accumulate in the cerebral cortex, hippocampus, and striatum of rats. However, why iron accumulates and in what oxidation state iron it accumulates in the brain of PS-exposed rats has not been well elucidated. In the present study, we investigated the influence of PS on the low molecular weight iron pool (LMWIP) in the rat brain. The results showed that: (1) PS significantly expanded LMWIP in the cerebral cortex, hippocampus, and striatum in rats; (2) PS caused derangement of pyramidal cells and reduced the layers of pyramidal CA1 and CA2 neurons; (3) PS exposure greatly lowered the expression of ferritin (Fn) and hephaestin (HP) in the rat cortex and hippocampus; and (4) PS decreased superoxide dismutase, glutathione peroxidase, and glutathione level and increased malondialdehyde level in the cerebral cortex, hippocampus, and striatum in rats. These results indicated that PS could expand LMWIP significantly, which may be attributed to PS-induced decrease in Fn, HP expression, and the subsequent reduction in iron storage and utilization, and expansion of LMWIP could in turn lead to aggravation of oxidative damage.     

Induction of apoptotic change in the rat hippocampus caused by ferric nitrilotriacetate

Abstract

Iron, a source of oxidative stress, plays a major role in the pathology of neurodegenerative disease. In Alzheimer's disease, the hippocampus is vulnerable to oxidative stress, leading to impairment in memory formation. In our previous study, a brain oxidative reaction was induced after intraperitoneal injection of ferric nitrilotriacetate (Fe-NTA). However, since only a small amount of iron reached the brain in the previous study, Fe-NTA was administered into the hippocampus using an osmotic pump in this study.

After continuous injection of Fe-NTA for 2 weeks, a high level of apoptotic change was induced in the hippocampus, in accordance with the iron localization. After injection for 4 weeks, the hippocampus was totally destroyed. A small amount of iron infiltrated into the cerebral cortex and the striatum, and deposition was observed at the choroid plexus and ependymal cells. However, no apoptotic reaction or clear tissue injury was observed in these areas. In addition, muscarinic acetylcholine receptors (M1, M2, and M4) were decreased in both the cortex and hippocampus while it increased in the striatum. Thus, the hippocampus is likely vulnerable to oxidative stress from Fe-NTA, and the oxidative stress is considered to bring the disturbance in the muscarinic acetylcholine receptors.     

Pyruvate Administration Reduces Recurrent/Moderate Hypoglycemia-Induced Cortical Neuron Death in Diabetic Rats

Recurrent/moderate (R/M) hypoglycemia is common in type 1 diabetes patients. Moderate hypoglycemia is not life-threatening, but if experienced recurrently it may present several clinical complications. Activated PARP-1 consumes cytosolic NAD, and because NAD is required for glycolysis, hypoglycemia-induced PARP-1 activation may render cells unable to use glucose even when glucose availability is restored. Pyruvate, however, can be metabolized in the absence of cytosolic NAD. We therefore hypothesized that pyruvate may be able to improve the outcome in diabetic rats subjected to insulin-induced R/M hypoglycemia by terminating hypoglycemia with glucose plus pyruvate, as compared with delivering just glucose alone. In an effort to mimic juvenile type 1 diabetes the experiments were conducted in one-month-old young rats that were rendered diabetic by streptozotocin (STZ, 50mg/kg, i.p.) injection. One week after STZ injection, rats were subjected to moderate hypoglycemia by insulin injection (10U/kg, i.p.) without anesthesia for five consecutive days. Pyruvate (500mg/kg) was given by intraperitoneal injection after each R/M hypoglycemia. Three hours after last R/M hypoglycemia, zinc accumulation was evaluated. Three days after R/M hypoglycemia, neuronal death, oxidative stress, microglial activation and GSH concentrations in the cerebral cortex were analyzed. Sparse neuronal death was observed in the cortex. Zinc accumulation, oxidative injury, microglial activation and GSH loss in the cortex after R/M hypoglycemia were all reduced by pyruvate injection. These findings suggest that when delivered alongside glucose, pyruvate may significantly improve the outcome after R/M hypoglycemia by circumventing a sustained impairment in neuronal glucose utilization resulting from PARP-1 activation.     

Erratum to: Corticosterone Induces Dysregulation of Iron Metabolism in Hippocampal Neurons In Vitro

Iron is required for neuronal function but in excess generates neurodegeneration. Although the iron homeostasis machinery in neurons has been described extensively, little is known about the influence of corticosterone on the iron homeostasis in neurons. In this study, we characterized the response of hippocampal neurons to a model of progressive corticosterone condition. We found that increasing extracellular corticosterone-induced iron accumulation killed a large proportion of neurons. Iron concentrations were significantly increased in the corticosterone-treated cells. In the hippocampal neurons, corticosterone decreased expression of ferritin and increased expression of transferrin receptor1 (TfR1), iron regulatory protein1 (IRP1), and divalent metal transporter 1. Corticosterone-induced elevation of IRP1 expression can cause increase of TfR1 and decrease of ferritin expression, which further leads to iron accumulation in hippocampal neurons and subsequently increases the oxidative damage of the neurons; it is indicated that corticosterone might be an important reason for iron deposition-caused neurodegenerative diseases.     

Corticosterone Induces Dysregulation of Iron Metabolism in Hippocampal Neurons In Vitro

Iron is required for neuronal function but in excess generates neurodegeneration. Although the iron homeostasis machinery in neurons has been described extensively, little is known about the influence of corticosterone on the iron homeostasis in neurons. In this study, we characterized the response of hippocampal neurons to a model of progressive corticosterone condition. We found that increasing extracellular corticosterone-induced iron accumulation killed a large proportion of neurons. Iron concentrations were significantly increased in the corticosterone-treated cells. In the hippocampal neurons, corticosterone decreased expression of ferritin and increased expression of transferrin receptor1 (TfR1), iron regulatory protein1 (IRP1), and divalent metal transporter 1. Corticosterone-induced elevation of IRP1 expression can cause increase of TfR1 and decrease of ferritin expression, which further leads to iron accumulation in hippocampal neurons and subsequently increases the oxidative damage of the neurons; it is indicated that corticosterone might be an important reason for iron deposition-caused neurodegenerative diseases.